Hematopoietic bone marrow cells participate in endothelial, but not epithelial or mesenchymal cell renewal in adult rats.

The extent to which bone marrow (BM) contributes to physiological cell renewal is still controversial. Using the marker human placental alkaline phosphatase (ALPP) which can readily be detected in paraffin and plastic sections by histochemistry or immunohistochemistry, and in ultrathin sections by electron microscopy after pre-embedding staining, we examined the role of endogenous BM in physiological cell renewal by analysing tissues from lethally irradiated wild-type inbred Fischer 344 (F344) rats transplanted (BMT) with unfractionated BM from ALPP-transgenic F344 rats ubiquitously expressing the marker. Histochemical, immunohistochemical and immunoelectron microscopic analysis showed that the proportion of ALPP(+) capillary endothelial cells (EC) profoundly increased from 1 until 6 months after BMT in all organs except brain and adrenal medulla. In contrast, pericytes and EC in large blood vessels were ALPP(-) . Epithelial cells in kidney, liver, pancreas, intestine and brain were recipient-derived at all time-points. Similarly, osteoblasts, chondrocytes, striated muscle and smooth muscle cells were exclusively of recipient origin. The lack of mesenchymal BM-derived cells in peripheral tissues prompted us to examine whether BMT resulted in engraftment of mesenchymal precursors. Four weeks after BMT, all haematopoietic BM cells were of donor origin by flow cytometric analysis, whereas isolation of BM mesenchymal stem cells (MSC) failed to show engraftment of donor MSC. In conclusion, our data show that BM is an important source of physiological renewal of EC in adult rats, but raise doubt whether reconstituted irradiated rats are an apt model for BM-derived regeneration of mesenchymal cells in peripheral tissues.

In-vivo generation of bone via endochondral ossification by in-vitro chondrogenic priming of adult human and rat mesenchymal stem cells.

BACKGROUND: Bone grafts are required to repair large bone defects after tumour resection or large trauma. The availability of patients' own bone tissue that can be used for these procedures is limited. Thus far bone tissue engineering has not lead to an implant which could be used as alternative in bone replacement surgery. This is mainly due to problems of vascularisation of the implanted tissues leading to core necrosis and implant failure. Recently it was discovered that embryonic stem cells can form bone via the endochondral pathway, thereby turning in-vitro created cartilage into bone in-vivo. In this study we investigated the potential of human adult mesenchymal stem cells to form bone via the endochondral pathway. METHODS: MSCs were cultured for 28 days in chondrogenic, osteogenic or control medium prior to implantation. To further optimise this process we induced mineralisation in the chondrogenic constructs before implantation by changing to osteogenic medium during the last 7 days of culture. RESULTS: After 8 weeks of subcutaneous implantation in mice, bone and bone marrow formation was observed in 8 of 9 constructs cultured in chondrogenic medium. No bone was observed in any samples cultured in osteogenic medium. Switch to osteogenic medium for 7 days prevented formation of bone in-vivo. Addition of ß-glycerophosphate to chondrogenic medium during the last 7 days in culture induced mineralisation of the matrix and still enabled formation of bone and marrow in both human and rat MSC cultures. To determine whether bone was formed by the host or by the implanted tissue we used an immunocompetent transgenic rat model. Thereby we found that osteoblasts in the bone were almost entirely of host origin but the osteocytes are of both host and donor origin. CONCLUSIONS: The preliminary data presented in this manuscript demonstrates that chondrogenic priming of MSCs leads to bone formation in vivo using both human and rat cells. Furthermore, addition of ß-glycerophosphate to the chondrogenic medium did not hamper this process. Using transgenic animals we also demonstrated that both host and donor cells played a role in bone formation. In conclusion these data indicate that in-vitro chondrogenic differentiation of human MSCs could lead to an alternative and superior approach for bone tissue engineering.

Marker tolerant, immunocompetent animals as a new tool for regenerative medicine and long-term cell tracking.

BACKGROUND: Immune-mediated rejection of labeled cells is a general problem in transplantation studies using cells labeled with any immunogenic marker, and also in gene therapy protocols. The aim of this study was to establish a syngeneic model for long-term histological cell tracking in the absence of immune-mediated rejection of labeled cells in immunocompetent animals. We used inbred transgenic Fischer 344 rats expressing human placental alkaline phosphatase (hPLAP) under the control of the ubiquitous R26 promoter for this study. hPLAP is an excellent marker enzyme, providing superb histological detection quality in paraffin and plastic sections. RESULTS: Transplantation of cells from hPLAP transgenic (hPLAP-tg) F344 rats into wild-type (WT) F344 recipients failed because of immune-mediated rejection. Here we show that this problem can be overcome by inducing tolerance to the marker gene by transplantation of bone marrow from hPLAP-tg F344 rats into WT F344 hosts after lethal irradiation, or by neonatal exposure of WT F344 rats to hPLAP-tg F344 cells. As proof-of-principle, we injected bone marrow cells (BMC) from hPLAP-tg rats into the knee joint of marker tolerant, bone marrow-transplanted WT rats, and found successful engraftment and differentiation of donor cells. In addition, hPLAP-tg BMC injected intravenously in neonatally tolerized WT F344 hosts could be traced in lymph nodes, 2 months post-injection. CONCLUSION: In combination with the excellent marker hPLAP, marker tolerant animals may open up new perspectives for all experiments requiring long-term histological tracking of genetically labeled cells.

Tracking mesenchymal stem cell contributions to regeneration in an immunocompetent cartilage regeneration model.

It is currently controversially discussed whether mesenchymal stem cells (MSC) facilitate cartilage regeneration in vivo by a progenitor- or a nonprogenitor-mediated mechanism. Here, we describe a potentially novel unbiased in vivo cell tracking system based on transgenic donor and corresponding immunocompetent marker-tolerant recipient mouse and rat lines in inbred genetic backgrounds. Tolerance of recipients was achieved by transgenic expression of an immunologically neutral but physicochemically distinguishable variant of the marker human placental alkaline phosphatase (ALPP). In this dual transgenic system, donor lines ubiquitously express WT, heat-resistant ALPP protein, whereas recipient lines express a heat-labile ALPP mutant (ALPPE451G) resulting from a single amino acid substitution. Tolerance of recipient lines to ALPP-expressing cells and tissues was verified by skin transplantation. Using this model, we show that intraarticularly injected MSC contribute to regeneration of articular cartilage in full-thickness cartilage defects mainly via a nonprogenitor-mediated mechanism.

Experimental Myocardial Infarction Upregulates Circulating Fibroblast Growth Factor-23.

Myocardial infarction (MI) is a major cause of death worldwide. Epidemiological studies have linked vitamin D deficiency to MI incidence. Because fibroblast growth factor-23 (FGF23) is a master regulator of vitamin D hormone production and has been shown to be associated with cardiac hypertrophy per se, we explored the hypothesis that FGF23 may be a previously unrecognized pathophysiological factor causally linked to progression of cardiac dysfunction post-MI. Here, we show that circulating intact Fgf23 was profoundly elevated, whereas serum vitamin D hormone levels were suppressed, after induction of experimental MI in rat and mouse models, independent of changes in serum soluble Klotho or serum parathyroid hormone. Both skeletal and cardiac expression of Fgf23 was increased after MI. Although the molecular link between the cardiac lesion and circulating Fgf23 concentrations remains to be identified, our study has uncovered a novel heart-bone-kidney axis that may have important clinical implications and may inaugurate the new field of cardio-osteology.

Histological assessment of cellular half-life in tissues in vivo.

The assessment of cellular half-life is of fundamental importance for cell biology and biomedicine. Here, we show that cellular half-life in tissues can be histologically measured under steady state conditions in vivo by analyzing the loss of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells over time after withdrawal of long-term BrdU labeling. To achieve efficient continuous cell labeling, we implanted BrdU-containing subcutaneous slow-release pellets into 12-month-old male Fischer 344 rats, delivering BrdU at a dose of 75 mg/kg per day over 1 (n=20) or 3 weeks (n=20). Four to five rats each were killed directly after the labeling or 1, 3, and 7 weeks post-labeling. Cellular half-life after withdrawal of BrdU was analyzed by nonlinear regression analysis of the labeling index, using a model of one-phase exponential decay. We initially validated our technique in the duodenum, where we determined a half-life of 2.4 days for crypt cells. Next, we applied this method to other tissues, and found a half-life of 2.2 weeks for cardiac endothelial cells, and of 5-6 days for pancreatic duct cells. In conclusion, we believe that this novel approach is an important step forward in the histological assessment of cellular half-life.

Role of endogenous bone marrow cells in long-term repair mechanisms after myocardial infarction.

Homing and regenerative potential of endogenous bone marrow cells (BMC) in myocardial infarction (MI) is a controversial issue. Using human placental alkaline phosphatase (hPLAP) as genetic marker for cell tracking, we examined the influx of bone marrow-derived cells during tissue repair after the induction of MI over a study period of 17 weeks in wild-type inbred Fischer 344 rats, lethally irradiated and reconstituted with bone marrow (BM) from transgenic F344 rats expressing hPLAP under the control of the ubiquitous R26 promoter. During the early phases of tissue repair, hPLAP-positive macrophages, endothelial cells, fibroblasts and also myofibroblast-like cells were recruited from BM. However, only some hPLAP-positive endothelial cells, fibroblasts and myofibroblast-like cells persisted until 17 weeks after MI. With the exception of a single cell, there was no evidence of BM-derived cardiomyocytes throughout the study. Rather, some local cardiac progenitor cells appeared to differentiate into cardiomyocytes in the peri-infarct regions. In conclusion, our data show that the inflammation-induced influx of BM-derived cells into the infarction area is restricted to leukocytes, endothelial cells, fibroblasts and myofibroblast-like cells. Our long-term analysis casts doubt on the hypothesis that circulating BM-derived mesenchymal precursor cells participate in cardiomyogenesis after MI.

Utility of human placental alkaline phosphatase as a genetic marker for cell tracking in bone and cartilage.

It was the aim of the current study to evaluate the utility of human placental alkaline phosphatase (hPLAP) as a genetic marker for cell tracking in bone and cartilage, using transgenic Fischer 344 rats expressing hPLAP under the control of the ubiquitous R26 promoter [F344-Tg(R26-hPLAP)]. hPLAP enzyme activity was retained during paraffin and methylmethacrylate (MMA) embedding, and was best preserved using 40% ethanol as fixative. Endogenous alkaline phosphatase activity could be completely blocked by heat inactivation in paraffin and MMA sections, allowing histochemical detection of hPLAP in the complete absence of background staining. In addition, sensitive detection of hPLAP was also possible using immunohistochemistry. F344-Tg(R26-hPLAP) rats demonstrated ubiquitous expression of hPLAP in hematopoietic bone marrow cells and stromal cells such as osteoblasts, osteocytes, and chondrocytes. Osteoclasts only weakly expressed hPLAP. In conclusion, hPLAP provides superb detection quality in paraffin and plastic sections, and constitutes an excellent genetic marker for cell tracking in hard and soft tissues.